Protocol

Phospho-Proteomics

Volume 1355 of the series Methods in Molecular Biology pp 275-290

Phosphoprotein Detection by High-Throughput Flow Cytometry

  • Johannes LandskronAffiliated withBiotechnology Centre, University of OsloK.G. Jebsen Centre for Cancer Immunotherapy, University of OsloCentre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo
  • , Kjetil TaskénAffiliated withBiotechnology Centre, University of OsloK.G. Jebsen Centre for Cancer Immunotherapy, University of OsloCentre for Molecular Medicine Norway, Nordic EMBL Partnership, University of OsloK.G. Jebsen Inflammation Research Centre, University of OsloDepartment of Infectious Diseases, University of Oslo Email author 

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Abstract

Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like Western blotting, relies on phospho-epitope-specific antibodies. In contrast to the latter, however, multidimensional and directly quantifiable data is obtained at the single-cell level allowing separate analysis of small cell populations in complex cellular mixtures. Furthermore, up to 30 phospho-specific antibodies or antibodies identifying other posttranslational modifications in combination with cell surface markers can be analyzed in a single experiment. Utilizing a technique called fluorescent cell barcoding that enables combination of up to 64 samples into one tube for multiplex analysis and later data deconvolution, phospho flow cytometry is turned into a medium- to high-throughput technology.

Key words

Flow cytometry Phospho flow Phospho-specific antibody Fluorescent cell barcoding Signaling